Hepatic ischemia/reperfusion (I/R) injury produces necrosis and apoptosis in a variety of clinical settings, including liver surgery, liver preservation for transplantation, veno-occlusive disease, hemorrhagic shock, heart failure and toxic liver disease. The mitochondrial permeability transition (MPT) is a key mechanism contributing to apoptotic and necrotic hepatocellular death after I/R injury. The overall goal of this project is to understand the molecular mechanisms promoting the MPT and MPT-dependent apoptosis and necrosis after I/R. We hypothesize that: 1) a convergence of cellular and mitochondrial changes including NAD(P)H oxidation, Ca2+ loading, reactive oxygen species (ROS) formation, glutathione depletion and alkalinization contribute to MPT-dependent apoptosis and necrosis after I/R;2) a combination of iron release from lysosomes (first hit) and mitochondrial superoxide/hydrogen peroxide generation (second hit) acts to initiate hydroxyl radical formation and peroxidation chain reactions to promote MPT onset after I/R;3) the same mechanisms causing I/R injury to cultured hepatocytes underlie hepatic I/R injury in vivo;and 4) the X-linked inhibitor of apoptosis protein (XIAP) decreases during ischemia and sensitizes hepatocytes to the action of death receptor ligands and MPT-dependent apoptosis after reperfusion. To evaluate these hypotheses, we will use hepatocytes from rats and genetically modified mice to 1) characterize the mechanistic roles of extra- and intramitochondrial alterations (Ca2+, ROS, NAD(P)H, pH, etc.) in MPT induction after I/R to cultured hepatocytes;2) evaluate the contribution of chelatable iron released from lysosomes in promoting the MPT and cell death after reperfusion;3) validate findings in cultured hepatocytes in a mouse model of hepatic I/R to show directly that hepatic I/R induces the MPT in vivo, that both apoptosis and necrosis follow the MPT in vivo, and that the major findings and conclusions from Specific Aims 1 and 2 extend to in vivo hepatic I/R;and 4) examine in vitro and in vivo l/R-induced apoptotic signaling in XIAP deficient and wildtype hepatocytes and in hepatocytes with adenoviral XIAP overexpression. The proposed work will combine a variety of experimental approaches, especially confocal and multiphoton microscopy of living hepatocytes labeled with parameter-indicating fluorophores and intravital multiphoton microscopy of livers in anesthetized mice. Our results will enhance the understanding of the molecular mechanisms underlying I/R injury to liver and lead to practical strategies to decrease injury and improve liver function after human hepatic ischemia in a variety of disease states.